RNA editing by adenosine deamination is a posttranscriptional mechanism for the regulation of gene expression and particularly widespread in mammals. A-to-I RNA editing regulates important functional properties of neurotransmitter receptor genes in the central nervous system by changing single codons in pre- mRNA. The deficiency or misregulation of editing has been implicated in the etiology of neurological diseases, such as epilepsy, amyotrophic lateral sclerosis, depression and tumor progression. Widespread A-to-I modification in the human transcriptome suggests additional roles for RNA editing and links it to other RNA processing phenomena, such as alternative splicing, siRNA mediated gene silencing and miRNA function. The long-term goal of these studies is to determine how RNA editing is regulated in vivo as an essential prerequisite for understanding its significance in gene regulation and the involvement of editing deregulation in disease mechanisms. Research on the mechanisms of epilepsy, neuromuscular disorders, and the molecular analysis of brain tumors has been identified by the NINDS as an important and pressing goal. The proposed research activities will determine the potency of putative regulatory mechanisms identified in preliminary studies to modulate A-to-I RNA editing activity. Using a reporter gene system that allows fast and quantitative analysis of cellular editing activity, candidate regulatory mechanisms will be characterized in human cells. The specific aims are to: 1. Determine the role of nuclear import in the regulation of intracellular RNA editing activity. Based on preliminary data showing differential interaction of editing enzymes with the family of nuclear localization signal (NLS) receptors, cellular transfection assays will be employed to define if intracellular RNA editing can be altered through selective overexpression or knocking- down of individual NLS receptor proteins. 2. Assess candidate molecular mechanisms for their ability to regulate A-to-I editing. Previous studies have suggested a number of regulatory mechanisms for RNA editing. A previously constructed and tested RNA editing reporter gene will be stably integrated into a human cell line and this cell line will subsequently be used to quantitatively measure the effect of such mechanisms on intracellular RNA editing activity. These studies will lead to a better understanding of how potential regulatory mechanisms influence cellular RNA editing activity. The quantitative and fast assay system will greatly accelerate the progress towards the proposed aims and promises to generate insights with high impact on future developments in the field. The research activities will involve extensive participation of graduate and undergraduate students in the laboratory. [unreadable] [unreadable] [unreadable]